Current Research journal of Biological Sciences 4(4): 372-380, 2012
ISSN: 2041-0778
© Maxwell Scientific Organization, 2012
Submitted: November 28, 2011
Accepted: December 27, 2011
Published: July 10, 2012
Response of Bread Wheat Genotypes to Immature Embryo Culture, Callus
Induction and Drought Stress
1
Parvin Elyasi, 1Ezatollah Farshadfar and 2Mostafat Aghaee
College of Agriculture, Razi University, Kermanshah, Iran
2
Seed and Plant Improvement Institute, Karaj, Iran
1
Abstract: In order to evaluate the response of twenty genotypes of bread wheat (Triticum aestivum L.) to callus
induction and in vitro drought stress. The immature embryos of wheat were used in a Completely Randomized
Design (CRD) with six replications for callus induction and a 20×2 factorial experiment based on CRD design
with three replications was carried out for response of genotypes to in vitro drought stress at the Agricultural
College of Razi University, Kermanshah, Iran during 2010-2011. Significant differences were observed among
the genotypes for Callus Growth Rate (CGR), Relative Fresh Weight Growth (RFWG), Relative Growth Rate
(RGR) and Percentage of Callus Induction (PCI) indicating the presence of genetic variability, different
responses of bread wheat genotypes to callus induction and possible selection of callus induction at in vitro
level using immature embryos. Mean comparison of the traits measured in callus induction showed that
genotypes 1 and 6 had the highest PCI (100%). Analysis of variance for CGR, RFWG and RGR, Relative Water
Content (RWC), Percent of Callus Chlorosis (PCC) and Proline Content (PC) exhibited significant differences
among the genotypes for all the characters in the stress condition (15% PEG). Screening drought tolerant
genotypes and in vitro indicators of drought tolerance using mean rank, standard deviation of ranks and biplot
analysis, discriminated genotypes (6), (19) and (1) as the most drought tolerant.
Keywords: Biplot analysis, embryo culture, in vitro indicators of drought tolerance, screening techniques
better use of water through the development of crop
varieties which need less water and are more tolerant to
drought (Shao et al., 2006; El-Shafey et al., 2009;
Mafakheri et al., 2010). Development of cultivars with
high yield is the main goal in water limited environments
but success has been modest due to the varying nature of
drought and the complexity of genetic control of plant
responses (Mirbahar et al., 2009). Since yield is a
complex trait and is strongly influenced by the
environment, severe losses can be caused by drought, a
stress common in most arid and semi arid areas.
Accordingly, drought tolerance is one of the main
components of yield stability and its improvement is a
major challenge to geneticists and breeders (Eid, 2009).
These efforts have been focused mostly on exploiting
high yield potential and genotype selection for
morphological, physiological and agronomic traits
indicative of drought tolerance in field conditions
(Dhanda et al., 2004).
Breeding for drought tolerance by selecting solely for
grain yield is difficult because the heritability of yield
under drought conditions is low, due to small genotypic
variance or due to the large variances in the genotypeenvironment interaction (Ludlow and Muchow, 1990;
Koszegi et al., 1996). Improvement of the wheat plant
itself gives a long-term avenue for raising its yield in the
INTRODUCTION
Cereal crops belonging to Graminae family
producing large edible grains which provide about onehalf of man,s food calories and a major portion of his
nutrient requirements (Jain, 2001). High adaptation of
bread wheat (Triticum aestivum L.) as well as its diverse
consumption in the human nutrition lead to be presented
as the most important cereal in the word, especially in the
developing countries and it can provides 20% food
resources of the world people (Farzi et al., 2010). Global
warming and concomitant increase in drought effected
areas limit plant production is also restricted by drought
exposed areas and this loss lead to considerable economic
and social problems because of its great importance on
human nutrition (Ilker et al., 2011). Water deficit the main
environmental constraint limiting cereal yield worldwide
and particularly within the Mediterranean basin, a
problem likely to become even worse in the future. Cereal
plants respond to drought through morphological,
physiological and metabolic modifications occurring in all
organs and therefore traits associated with improved
performance under water limited conditions or improved
survival to extremely low water availability are diverse
(Slafer et al., 2005). One possible way to ensure future
food needs of an increasing world population involves the
Corresponding Author: Ezatollah Farshadfar, College of Agriculture, Razi University, Kermanshah, Iran
372
Curr. Res. J. Biol. Sci., 4(4): 372-380, 2012
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field. Thus, under stressful environments, yield per se is
not always the most suitable or easiest selection trait and
an approach based on the evaluation and incorporation of
physiological traits into a potentially high-yielding
genotype may improve its adaptability and thus its
response to environmental variability (Steven et al., 1990;
Blum, 2005). Much attention is shifted towards crop
improvement programs. On of such biotechnological
techniques is the plant tissue culture. Tissue culture
techniques are becoming increasingly popular as an
alternative means of plant vegetative propagation, mass
production of chemicals, and genetic engineering
(Shah et al., 2009). Resent progress in genetic
manipulation of plant cells has opened new possibilities
in crop improvement. Callus culture are used as an in
vitro technique for biochemical and physiological studies
in response to stress at the cellular level (Liu et al., 2006).
Many researchers have used the in vitro culture of cells on
media supplemented with PEG to study the mechanisms
of drought tolerance and to utilize the somaclonal
variation, as a source of variability to improve the drought
tolerance (El-Shafey et al., 2009). Various osmotic agents
have been employed in appropriate nutrient media to
screen germplasm in vitro for drought tolerance. Although
specific in vitro methods vary with plant types being
screened, researchers have been able to control the
drought environmental more precisely using in vitro or
artificial selection techniques (Maruyama et al., 2008; He
et al., 2009; Srinivasan et al., 2010). Polyethylen Glycol
(PEG) of high molecular weights, have long been used to
stimulate water stress in plants (Ruf et al., 1967;
Kaufmann and Eckard, 1971; Corchete and Guerra,
1986). PEG of high molecular weight is a non penetrating inert osmoticum lowering the water potential
of nutrient solutions without being taken up or being
phytotxic (Lawlor, 1970).
Osmotic solutions of NaCl, mannitol/sorbitol and
Polyethylene Glycol (PEG) have been used as in vitro
stress factors for selecting salt- and drought-tolerant
genotypes in screening procedures for seed germination
of wheat (Almansouri et al., 2001), sunflower (Punia and
Jain, 2002) and potato (Gopal and Iwama, 2007).
The objectives of the present investigations were to:
C
C
C
WC - 5047
WC - 4530
WC - 4780
WC - 4566
WC - 47360
WC - 4640
WC - 47456
WC - 47628
WC - 47367
WC - 47399
WC - 47636
WC - 4584
WC - 46697 - 11
WC - 4823
Pishtaz
WC- 47341
WC - 47379
WC - 4931
WC - 47381
WC - 5053
kindly provided from Seed and Plant Improvement
Institute of Karaj, Iran to callus induction and in vitro
drought stress. A Completely Randomized Design (CRD)
with six replications was used for callus induction and a
20×2 factorial experiment based on CRD design with
three replications was carried out for response of
genotypes to in vitro drought stress at the Agricultural
College of Razi University, Kermanshah, Iran during
2010-2011.
The genotypes were exposed to different
concentrations of PEG 6000 (Merck, Germany) (0 as
control and 15%) for 14 days. The growing morphogenic
calli derived from immature embryos were also exposed
to Murashige and Skooge (1962) medium containing
different concentrations of PEG (0 and 15%). Spikes were
harvested from main tillers 14 days post-anthesis. Spikes
rinsed twice with water then were surface-sterilized in
70% (v/v) ethanol for 1 min, rinsed twice with sterile
distilled water, incubated further in commercial bleach
(5% sodium hypochlorite) for 10 min and rinsed several
times in sterile distilled water. All the operations and
inoculation were performed under strict aseptic conditions
in a laminar airflow cabinet. Immature embryos were
aseptically dissected from the seeds and placed scutellum
up on MS medium supplemented with 30 g/L sucrose and
was adjusted to PH 5.7, solidified with 8g/L agar and 2.5
mg/L 2,4-dichlorophenoxy acetic acid (2,4-D)(Merck,
Germany). The medium was autoclaved at 121ºC for 20
min and incubated at 25ºC for 28 days in growth chamber
and in the darkness. Callus was maintained by subculturing every 21-28 days on the same MS medium. In
drought stress conditions the cultures were kept in an
incubator without any light. The following callus
characteristics were measured under stress conditions:
Screen bread wheat genotypes for drought tolerance
under in vitro condition
Evaluate the ability of genotypes to induce callus
using immature embryo culture
Screening in vitro indicators of drought tolerance
MATERIALS AND METHODS
In order to evaluate the response of twenty genotypes
of bread wheat (Triticum aestivum L.) namely:
373
Curr. Res. J. Biol. Sci., 4(4): 372-380, 2012
Percentage of Callus Induction (PCI): PCI was
evaluated 4 weeks (suitable for sub-culturing) after
embryo culture in Petri dishes as: (Arzani and Mirodjagh,
1999) (number of seeds producing callus)/(number of
seeds plated in Petri dishes).
where, W0 is the weight of callus before treatment and W1
the final weight of callus after two weeks of treatment.
Callus growth index was calculated for two levels of PEG
(0 and 15%) and the average of two levels was used for
calculation.
Relative Fresh Weight Growth (RFWG): RFWG =
[(W2-W1)]/W1 (Chen et al., 2006) where W1 and W2 are
the initial weight of callus before and after four weeks,
respectively.
Relative tolerance (Rt%): percentage of Rt% was
calculated for each genotype using the following formula
(Abdelsamad et al., 2007):
Relative Growth Rate (RGR): RGR = [LnW2-LnW1]/GP
(Birsin and Ozgen, 2004) where W1 and W2 are the initial
and final weight of callus and GP is the growth period,
respectively. The time interval between two consecutive
measurements was 21 days.
Rt % = [(value under stress)/ (value under nonstress)] × 100
Reduction percentage (R%): R% was calculated for the
two stress (15%) and non-stress level (0) using the
following formula (Abdelsamad et al., 2007):
(value under 15% stress level n- value at 0% stress level).
Callus Growth Rate (CGR): CGR (mm/day) of cultured
embryos on MS medium were measured at 7, 14, 21 and
28 days, respectively after transferring calli to medium.
CGR was calculated using the following formulas
(Compton, 1994):
Proline Content (PC): Extraction and estimation of free
praline content were done according to the procedure
described by Errabii et al. (2007).
CGR1 = d7/7, CGR2 = d14 /7, CGR3 = d21/7, CGR4 = d28/7
CGR = (CGR1+ CGR2 + CGR3 + CGR4) / 4
Statistical analysis: Analysis of variance, mean
comparison using Duncan,s Multiple Range Test
(DMRT), correlation analysis between mean of the
characters measured and principal component analysis
(PCA), based on the rank correlation matrix were
performed by MSTAT-C, SPSS ver. 16 and
STATISTICA ver. 8. Standard Deviation of Ranks (SDR)
was measured as:
where d7, d14, d21, d28, respectively were diameter of callus
in days 7, 14, 21 and 28, respectively. Diameter of callus
was calculated as:
Diameter of callus = DC =%length×width
Percentage of Callus Chlorosis (PCC): PCN was
determined visually as percentage of necrotic callus, 16
days after moving callus to the PEG containing medium.
m
 (R
S 
2
i
Relative Water Content (RWC): callus samples of
known fresh weight were dried in an oven set at 700C for
24 h and RWC was calculated by following formula
(Errabi et al., 2006):
j 1
ij
 Ri . ) 2
l1
where, Rij is the rank of in vitro drought tolerance
indicator and Ri. is the mean rank across all in vitro
drought tolerance indicators for the ith genotypes and
SDR = (S2i)0.5 (Arzani and Mirodjagh, 1999).
RWC = [(FW-DW)/DW]×100
RESULTS AND DISCUSSION
where, FW and DW are the callus fresh and dry weights,
respectively.
Callus induction: Highly significant differences (p<0.01)
were observed among the genotypes for CGR, RFWG,
RGR and PCI, respectively indicating the presence of
genetic variability, different responses of genotypes to
callus induction and possible selection of callus induction
in bread wheat using immature embryos of wheat
(Table 1). Immature embryo culture supplement with 2,
4-D gave good callus growth (Shan et al., 2000;
El-Sherbeny et al., 2001). Immature embryos of 1.0-1.5
mm,14 d after anthesis were cultured on MS medium
supplemented with 1.5 or 2.0 mg/L 2, 4-D and found that
90-100% of these embryos formed callus (Arun et al.,
In Vitro Tolerance (INTOL): INTOL was calculated
according to the following formula (Al-Khayri and AlBahrany, 2004):
INTOL = RGRtreatment / RGRcontrol
where, RGR = relative growth rate and was measured by
the formula of Birsin and Ozgen (2004).
Callus Growth Index (CGI): or increasing value of
callus fresh weight was calculated as: CGI = (W1-W0)/W0
(Abdelsamad et al., 2007):
374
Curr. Res. J. Biol. Sci., 4(4): 372-380, 2012
Table 1: Analysis of variance for callus induction traits in bread wheat
MS
--------------------------------------S.O.V
df
CGR
RFWG
RGR
PCI
Genotypes 19
0.003**
0.826**
0.009**
0.029**
Error
100
0.001
0.058
0.001
0.002
CV%
3.11
14.60
12.11
2.59
**: Significant at 1% level of probability
Table 2: Mean comparison for callus traits using DMRT*
Genotype CGR
RFWG
RGR
1
0.1964abc 0.9124h
0.0309g
2
0.262abc
1.5021fgh
0.0395efg
3
0.2069abc 1.2092gh
0.0416defg
4
0.183ab
3.1033abc
0.0663ab
5
0.1998abc 3.1919abc
0.0687ab
6
0.2246abc 1.3232fgh
0.0386efg
7
0.2194abc 2.4822cde
0.0546bcde
8
0.2383abc 1.9224defg
0.0495bcdef
9
0.3084d
4.2225a
0.0763a
10
0.2023abc 2.065def
0.053bcde
11
0.2284abc 2.0867def
0.0523bcde
12
0.2198abc 0.9387efg
0.0469cdefg
13
0.2454bc
2.7803bcd
0.0627abc
14
0.2550c
3.898ab
0.0747a
15
0.1965abc 1.8204defg
0.0492bcdef
16
0.2447bc
2.4247cde
0.0582abcd
17
0.2489b
c3.2705abc
0.0687ab
18
0.1754a
0.4671i
0.0179h
19
0.2422abc 0.9216h
0.0296g
20
0.2295abc 1.0268h
0.0332fg
*: Common letters indicate no significant difference
induction traits (Table 2) obviously revealed that culture
response was greatly influenced by the wheat genotypes
and also emphasized a profound effect of genotypes on
callus induction capacity, which is in agreement with
reports of callus induction in durum wheat (Ozgen et al.,
1996; Bommineni and Jauhar, 1996) and in bread wheat
(Hess and Carman, 1998). The importance of genotype in
determining the in vitro response of wheat tissues has
been recognized and the efficiency of callus induction,
callus growth rate and plant regeneration frequency have
all been reported to be genotype dependent (Yadav et al.,
2000; Yadava and Chawla, 2001; Schween and Schwnkel,
2003). Birsin and Ozgen (2004) reported that the
genotype effects on callusing ability from triticale mature
embryo cultures. Shah et al. (2009) exhibited significant
differences between and among wheat cultivars for callus
induction response and the callus induction was found to
be genotype-dependent. In general, callus induction used
as on efficient character for assessment of culture
responses from mature embryo in wheat genotypes. The
callus fresh weight is provided a more concise
quantitative character for the development rate of callus.
PCI
100.00a
80.00bcde
83.33bcde
75.00de
76.60bcde
100.00a
50.00g
88.33ab
88.33ab
85.00bcd
80.00bcde
61.66e
76.66cde
78.33bcde
78.33bcde
81.66bcde
63.33f
83.33bcde
93.33a
86.60bc
Effect of drought stress on the characters: Analysis of
variance for Callus Growth Rate (CGR), Relative Fresh
Weight Growth (RFWG), Relative Growth Rate (RGR),
Relative Water Content (RWC), Percent of Callus
Chlorosis (PCC) and Proline Content (PC) indicated
highly significant differences (p<0.01) among the
genotypes for all the characters in the stress condition
(15%) (Table 3). The analysis of variance also showed
significant differences among levels of (0, 15%) PEG
concentration for traits CGR, RFWG, RGR, RWC, PCC
and genotypeWdrought interaction for CGR, RGR, RWC
and PCC, respectively. The result obtained from
comparison of means revealed that the highest amount of
CGR, RFWG, RGR, RWC and PC, respectively belonged
to genotypes no.14, 6, 6, 6 and 19, respectively. While the
lowest amount of CGR, RFWG, RGR, RWC and PC,
respectively was attributed to genotypes no. 5, 7, 7, 7 and
12, respectively (Table 4). The highest PCC and the
lowest PCC were related to genotypes 17 and 1,
respectively. The results indicated that CGR, RFWG,
RGR and RWC decreased in the stress condition (%15
PEG level) as compared with non-stress condition (0%
1994). Arzani et al. (1999) reported that there were
significant differences among cultivars for potential of
regeneration from immature embryo and the Fresh Weight
Growth of callus (FWG) distinguished cultivars more than
callus induction frequency did for callus induction
evaluation. Solid MS medium was optimum for immature
embryo culture (Al-khayri and Al-Bahrany, 2000; Delport
et al., 2000; Mendoza and Kaeppler, 2002) of wheat
supplement with different combinations of plant growth
regulators.
Mean comparison of traits in callus induction: Mean
comparison of the traits measured in callus induction
showed that genotypes, 1 and 6 had the highest PCI
(100%). The highest amount of CGR, RFWG and RGR
belonged to genotype no.9. While the lowest amount of
CGR, RFWG and RGR was attributed to genotypes no.18,
1 and 18, respectively (Table 2). The results of callus
Table 3: Analysis of variance for mature embryos callus characters under stress condition
MS
-------------------------------------------------------------------------------------S.O.V
d.f
CGR
RFWG
RGR
RWC
PCC
PC
Genotype(G)
19
0.011**
0.010**
0.001**
0.005**
0.115**
0.830**
Drought(D)
1
0.012**
0.227**
0.016*
0.293**
2.095**
1.021 ns
DWG
19
0.002**
0.007ns
0.0002**
0.005**
0.027**
0.483 ns
Error
80
0.001
0.004
0.0002
0.002
0.005
0.329
CV%
3.18
6.86
5.42
2.07
5.15
2.32
Ns, *,**: Non-significant, significant at 0.05 and significant at 0.01 level of probability, respectively.
375
Curr. Res. J. Biol. Sci., 4(4): 372-380, 2012
Table 4: Mean comparison of the traits measured in stress condition (p<0.01)*
Genotype
CGR
RFWG
RGR
1
1.27bcd
0.3364a
0.0159abc
2
1.52a
0.2888a
0.0139abc
3
1.33abc
0.4986a
0.0217ab
4
1.13ef
-0.0029abc
-0.0015cde
5
1.08fg
0.1029abc
0.00175 bcde
6
1.55a
0.5000a
0.0237a
7
1.58abc
-0.1278c
-0.0117e
8
1.59a
0.1886a
0.0103abc
9
1.37abc
0.4384a
0.0212ab
10
1.45ab
0.3435a
0.0163abc
11
1.60a
0.1790a
0.0085abcd
12
1.34abc
-0.0086abc
-0.0022cde
13
1.48a
0.2375a
0.0095abcd
14
1.74abc
0.4706abc
0.0172abc
15
1.18de
0.2373a
0.0109abc
16
1.61a
0.2363ab
0.0096abcd
17
1.25cd
-0.098bc
-0.0093de
18
1.0g
0.1326a
0.0072abcd
19
1.73abc
0.4853a
0.0228a
20
1.22de
0.3046a
0.0150abc
*: Common letters indicate no significant difference
RWC
83.20d
80.98abc
82.89ab
82.09ab
85.95a
88.77a
71.34ab
86.22a
83.82a
82.91ab
84.15a
82.48ab
81.64abc
75.12cd
80.26abc
82.74ab
74.20bcd
83.51a
83.62a
83.39a
PCC
16.14g
22.44f
21.30f
32.01cde
33.03bcd
21.71g
46.01a
21.69ef
19.99f
27.56bcd
31.88bc
40.86a
30.26bcd
32.61b
31.96bc
27.45bcd
48.00a
28.75bcd
21.72g
25.02de
PC
5.02ab
3.80cd
4.55bcd
2.59cd
3.24cd
6.35a
2.06bcd
4.20bcd
4.11bcd
3.35bcd
3.29bcd
1.51abc
2.59cd
2.82cd
3.99cd
4.31bcd
1.80bcd
4.29bcd
7.54abc
3.07d
Table 5: Mean comparison of in vitro indicators of drought tolerance under stress (15% PEG) and non-stress (0 % PEG) using immature embryo
culture (p<0.01)
Drought
CGR
RFWG
RGR
RWC
PCC
PC
0
1.46a
0.4577a
0.0220a
90.63a
19.76a
3.61a
15
1.35b
0.0166b
-0.0019b
73.09b
36.82b
5.58b
*: Common letters indicate no significant difference
manitol concentration for callus survival and regeneration
ability from immature embryos of wheat. Hamdy and
Aref (2002) examined the immature embryo culture of
maize for improving drought tolerance in January 25,
cultivars and reported that analysis of variance revealed
highly significant differences between the tested
genotypes as well as between the different levels of
drought (PEG concentration) for all studied characters.
Early works of Singh et al. (1972) displayed a significant
positive correlation between drough resistance and proline
accumulation in barley. Since then, a number of workers
have reported enhanced accumulation of proline content
in different plants (Szegletes et al., 2000;
Chandrasekar et al., 2000; Deora et al., 2001). Al-khayri
and Al-Bahrany (2000) examined the response of palm
(Phoenix dactylifera L.) calli to water stress. Callus
growth, water content and proline accumulation were
assessed. They showed that increasing water stress caused
a progressive reduction in growth as expressed in callus
fresh mass, relative growth rate and index of tolerance.
Abdulaziz and Al-Bahrany (2002) studied the callus to
varing degree of Polyethylene Glycol (PEG)-induced
water stress.They studied callus growth, water content and
prolin accumulation. Their results indicated that
increasing water stress induced by increasing
concentration of PEG caused a progressive reduction in
callus fresh weight. Significant reduction in callus weight
was recorded in response to 50g/L PEG. increasing with
a progressive reduction in callus water content, which
caused increase in proline accumulation reaching
PEG. Level). PC and PCC were increased in %15 PEG
level as compared with 0% PEG level (Table 5).
In vitro indicators of drought tolerance: Callus Growth
Index (CGI) displayed remarkable differences among the
genotypes in the means of increasing value of selected
calli. Genotype no.6 showed the highest callus increasing
value (Table 6). The highest amount of relative tolerance
(Rt%) in the induced drought stress condition was
attributed to genotype no.6 (Table 6), while the lowest
amount of reduction percentage (R%) from 0.0 to 15%
PEG belonged to genotype no.6 and the highest amount of
R% was shown by genotype no.14 (Table 6). The amount
of callus growth was expressed as in vitro tolerance
(INTOL) to eliminate inherent differences associated with
the Relative Growth Rate (RGR) of the genotypes in
response to induced drought stress by PEG. Based on
INTOL genotype no. 6 exhibited the highest INTOL
(Table 6). With regard to callus (resulted from immature
embryos) increasing value, percentage of relative
tolerance (Rt%), the amount of reduction percentage (R%)
and INTOL genotype no. 6 was selected as the most
drought tolerant at in vitro condition (Table 6).
The increasing value of proline concentration during
stress condition has been suggested as an osmoticum, a
desiccation-protectant, a sink for nitrogen and reducing
power during stress or a source of nitrogen and reducing
power during recovery from stress (Steven et al., 1990).
Abdel-Ghany et al. (2004) expressed that there were
highly significant interactions between cultivars for
376
Curr. Res. J. Biol. Sci., 4(4): 372-380, 2012
Table 6: (I): Ranks (R), ranks mean ( R ) and standard deviation of ranks (SDR) of in vitro indicators of drought tolerance using immature embryo
culture
Genotype no CGR
R
RFWG
R
RGR
R
RWC
R
INTOL
R
PCC
R
1
1.27
14
0.3364
7
0.01590
7
83.20
9
0.5665
3
16.14
1
2
1.52
8
0.2888
2
0.01390
9
80.98
16
0.0028
9
22.44
7
3
1.33
13
0.4986
9
0.02170
3
82.89
11
0.2840
6
21.30
3
4
1.13
18
-0.0029
17
-0.00150 17
82.09
14
-1.2800
18
32.01
15
5
1.08
19
0.1029
16
0.00175
16
85.95
3
-0.8394
17
33.03
17
6
1.55
7
0.5000
1
0.02370
1
88.77
1
0.8809
1
21.71
5
7
1.58
6
-0.1278
20
-0.01170 20
71.34
20
-6.8750
20
46.01
19
8
1.59
5
0.1886
13
0.01030
11
86.22
2
0.3464
4
21.69
4
9
1.37
11
0.4384
5
0.02120
4
83.82
5
0.3198
5
19.99
2
10
1.45
10
0.3435
6
0.01630
6
82.91
10
0.0900
8
27.56
10
11
1.60
4
0.1790
14
0.00850
14
84.15
4
-0.0502
11
31.88
13
12
1.34
12
-0.0086
18
-0.00220 18
82.48
13
-1.3700
19
40.86
18
13
1.48
9
0.2375
10
0.00950
13
81.64
15
-0.3003
15
30.26
12
14
1.74
1
0.4706
4
0.01720
5
75.12
18
-0.2013
14
32.61
16
15
1.18
17
0.2373
11
0.01090
10
80.26
17
-0.0641
12
31.96
14
16
1.61
3
0.2363
12
0.00960
12
82.74
12
-0.0252
10
27.45
9
17
1.25
15
-0.0980
19
-0.00930 19
74.20
19
-3.5600
16
48.00
20
18
1.01
20
0.1326
15
0.00720
15
83.51
7
-0.0649
13
28.75
11
19
1.73
2
0.4853
3
0.02280
2
83.62
6
0.6618
2
21.72
6
20
1.22
16
0.3046
8
0.01500
8
83.39
8
0.1278
7
2502
8
Table 6: continued
Genotype no
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
PC
5.02
3.80
4.55
2.59
3.24
6.35
2.06
4.20
4.11
3.35
3.29
1.51
2.59
2.82
3.99
4.31
1.80
4.29
7.54
3.07
R
3
10
4
17
13
2
18
7
8
11
12
20
16
15
9
5
19
6
1
14
CGI
0.1857
0.0424
0.0459
-0.1926
-0.2904
0.5061
-0.3148
0.0401
0.1417
0.0423
0.0113
-0.2194
-0.0042
-0.0346
-0.0529
-0.1476
-0.3537
-0.2345
0.2613
0.0592
R
3
7
6
15
18
1
19
9
4
8
10
16
11
12
13
14
20
17
2
5
Rt%
90.92
66.03
59.77
67.69
51.84
98.70
64.00
81.83
68.46
65.73
74.09
64.29
54.55
47.37
64.62
70.69
58.48
59.64
83.51
66.93
R
2
10
15
8
19
1
14
4
7
11
5
13
18
20
12
6
17
16
3
9
significant increase over the control. Abdelsamad et al.
(2007) declared that significant differences of genetic
responses were observed for the four wheat genotypes at
10 and 20% PEG for callus induction, callus fresh weight,
growth index, relative water content and relative tolerance
percentage. El-Shafey et al. (2009) indicated that osmotic
stress due to PEG application highly significantly
decreased the fresh weight of the non irradiated rice calli
as well as irradiated once in response to 10 and 15 %
PEG, as compared with the control.
R%
0.93
5.94
4.82
3.34
8.46
0.21
6.13
3.44
4.86
5.90
5.91
4.76
9.83
14.56
3.23
5.05
5.38
1.59
3.41
4..06
R
2
16
10
5
18
1
17
7
11
14
15
9
19
20
4
12
13
3
6
8
Sum
51.00
101.00
73.00
144.00
156.00
21.00
173.00
66.00
62.00
94.00
102.00
156.00
138.00
125.00
119.00
95.00
177.00
123.00
33.00
91.00
R
5.10
10.10
7.30
14.40
15.60
2.10
17.30
6.60
6.20
9.40
1.20
15.60
13.80
12.50
11.90
9.50
17.70
12.30
3.30
9.10
SDR
4.09
3.28
4.62
4.42
4.76
2.13
4.39
3.50
3.01
2.40
4.31
3.62
3.35
6.86
3.84
3.65
2.35
5.43
1.94
3.31
can be assigned to one group only (Khodadadi et al.,
2011). The relationships among different indices are
graphically displayed in a biplot of PCA1 and PCA2
(Fig. 1). The PCA1 and PCA2 axes which justify 77.74%
of total variation, mainly distinguish the indices in
different groups. One interesting interpretation of biplot
is that the cosine of the angle between the vectors of two
indices approximates the correlation coefficient between
them. The cosine of the angles does not precisely translate
into correlation coefficients, since the biplot does not
explain all of the variation in a dataset. Nevertheless, the
angles are informative enough to allow a whole picture
about the interrelationships among the in vitro indices
(Yan and Kang, 2003). Rt% and RWC we refer to group
1 = G1 indices which introduce genotype No. 6 as drought
tolerant. The PCs axes separated PC, CGI and INTOL in
a single group (G2) that identify genotypes No. 19, 6 and
Screening in vitro indicators of drought tolerance: To
better understand the relationships, similarities and
dissimilarities among the in vitro indicators of drought
tolerance, Principal Component Analysis (PCA), based on
the rank correlation matrix was used. The main advantage
of using PCA over cluster analysis is that each statistics
377
Curr. Res. J. Biol. Sci., 4(4): 372-380, 2012
they are recommended for crossing and genetic analysis
of drought tolerance using diallel mating design or
generation mean analysis and also for the QTLs
(quantitative trait loci) mapping and marker assissted
selection. Genotypes (15 = Pishtaz ), (10 = WC-47399),
(9 = WC-47367) and (3 = WC-4780) , respectively were
distinguished as semi-tolerant genotypes. The same
procedures have been used for screening quantitative
indicators of drought tolerance in wheat (Mohammadi et
al., 2011b), in maize (Farshadfar and Sutka, 2002) and in
rye (Farshadfar et al., 2003).
1.0
PCA 2 : 17.78%
0.5
0.0
-0.5
RT G 1
RCW
PC
CGI INTOL G 2
PGR
PFWG
G3
PCC
G6
G4
CGR
G5
P%
ACKNOWLEDGMENT
-1.0
-1.0
-0.5
0.0
PCA 1 : 59.96%
0.5
The authors express their appreciations to the Iran
National Science Foundation for providing financial
support for this research project (code number =
88002345).
1.0
Fig. 1: Biplot analysis of in vitro indicators of drought tolerance
using immature embryo culture
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